As the most important inhibitory neurotransmitter in the brain, a detailed understanding of the implications of gamma-aminobutyric acid (GABA) release remains elusive. The measurement of GABA concentrations is a difficult process. Microdialysis is the current standard for GABA sampling in the brains of freely moving animals, but suffers from low temporal resolution and the need for labor intensive analysis methods. By contrast, the direct sensing of GABA, by modalities including biosensors, provides second-by-second temporal resolution, without the need for additional post-analysis. However, biosensors and other monitoring devices, require an enzyme to process the analyte of interest. The state-of-the-art for the enzymatic conversion of GABA into a transducible signal is the sequential activity of three separate enzymes or antibodies entrapped within nanoparticles. For CNS and systemic GABA sensing applications, a single GABA oxidase enzyme is necessary. No such oxidase enzyme for GABA is currently available. To develop this enzyme, Pinnacle will team with an interdisciplinary group of two leading scientists at the University of Kansas. Professor Mark Richter, is an expert in protein engineering and protein folding, and Dr. Philip Gao, is the Director of the Protein Production Core Facility. This team has already cloned, expressed purified and characterized an oxidase enzyme (wt-pUUB-Ox) with some GABA activity. During Phase I, we will use this oxidase enzyme as a starting scaffold to evolve a true GABA oxidase enzyme. At the end of Phase 1, we will have an oxidase enzyme with a 10x - 50x improvement in GABA activity relative to wt-pUUB-Ox, and a clear path for Phase II to oxidase activity and stability suitable for the specific measurement of physiologically relevant GABA concentrations. This evolved GABA oxidase enzyme will, in a single reaction step, oxidize GABA to produce hydrogen peroxide as a byproduct. The GABA oxidase enzyme can be used as the basis for new monitoring paradigms that would otherwise be impossible. By the end of Phase II, two commercially available products will be available. First, a GABA biosensor for real-time measurement of physiologically relevant levels of GABA in the brain for preclinical models and second, a GABA oxidase enzyme for use in a variety of diagnostic and point-of-care devices.